Project Summary Kidney cancer incidence has been increasing steadily for the past several decades, although the reasons for this are unclear. The VHL tumor suppressor gene was identified as a germline mutation in patients at risk for clear cell renal cell carcinoma (ccRCC), which accounts for approximately 85% of all kidney cancers. More importantly, inactivating VHL mutations also play major roles in sporadic renal cell cancer. Work from many laboratories show that the pVHL- associated complex has E3 ubiquitin ligase activity and VHL loss leads to hypoxia inducible factor ? (HIF-?, including HIF1? and HIF2?) accumulation. While HIF1? serves mainly as a tumor suppressor in kidney cancer, HIF2? stabilization, as a result of pVHL loss, is sufficient and necessary for promoting kidney tumor growth. Therefore, HIF2? serves as an important therapeutic target in kidney cancer related to VHL loss. While HIF2? can undergo ubiquitination and degradation mediated by pVHL E3 ligase complex, it remains unclear whether HIF2? can also be regulated by deubiquitination pathways. Recently, we performed a deubiquitinase (DUB) siRNA screening (including all of known 96 DUB family members) in kidney cancer cells and identified OTUB1 as a specific deubiquitinase that regulate HIF2? protein levels in kidney cancer. We hypothesize that OTUB1 can serve as a novel therapeutic target in kidney cancer by deubiquitinating HIF2?. In specific aim 1, we will determine the mechanism by which OTUB1 regulates HIF2? protein stability in kidney cancer. In specific aim 2, we will determine the functional role of OTUB1 in kidney cancer in vitro and in vivo. Successful completion of this proposal will motivate the development of specific OTUB1 inhibitors that benefit kidney cancer patients.